Fluorescence assay of the interaction between hemoglobin and the cytoplasmic domain of erythrocyte membrane band 3

Martiana Florenta Sega, Haiyan Chu, John A. Christian, Philip S. Low

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Oxygen tension has emerged as a potent regulator of multiple erythrocyte properties, including glucose metabolism, cell volume, ATP release, and cytoskeletal organization. Because hemoglobin (Hb)1 binds to the cytoplasmic domain of band 3 (cdb3) in an oxygen dependent manner, with deoxyHb exhibiting significantly greater affinity for cdb3 than oxyHb, the deoxyHb-cdb3 interaction has been hypothesized to constitute the molecular switch for all O2-controlled erythrocyte processes. In this study, we describe a rapid and accurate method for quantitating the interaction of deoxyHb binding to cdb3. For this purpose, enhanced green fluorescent protein (eGFP) is fused to the COOH-terminus of cdb3, and the binding of Hb to the NH2-terminus of cdb3-eGFP is quantitated by Hb-mediated quenching of cdb3-eGFP fluorescence. As expected, the intensity of cdb3-eGFP fluorescence decreases only slightly following addition of oxyHb. However, upon deoxygenation of the same Hb-cdb3 solution, the fluorescence decreases dramatically (i.e. confirming that deoxyHb exhibits much greater affinity for cdb3 than oxyHb). Using this fluorescence quenching method, we not only confirm previously established characteristics of the Hb-cdb3 interaction, but also establish an assay that can be exploited to screen for inhibitors of the sickle Hb-cdb3 interaction that accelerates sickle Hb polymerization.

Original languageEnglish (US)
Pages (from-to)266-271
Number of pages6
JournalBlood Cells, Molecules, and Diseases
Volume55
Issue number3
DOIs
StatePublished - Oct 1 2015

Fingerprint

Erythrocyte Membrane
Hemoglobins
Fluorescence
Sickle Hemoglobin
Erythrocytes
Oxygen
Cell Size
Polymerization
Adenosine Triphosphate
Glucose
enhanced green fluorescent protein

Keywords

  • Anion exchanger 1
  • FRET analysis
  • Hemoglobin
  • Protein structure
  • Red blood cells

ASJC Scopus subject areas

  • Molecular Medicine
  • Hematology
  • Molecular Biology
  • Cell Biology

Cite this

Fluorescence assay of the interaction between hemoglobin and the cytoplasmic domain of erythrocyte membrane band 3. / Sega, Martiana Florenta; Chu, Haiyan; Christian, John A.; Low, Philip S.

In: Blood Cells, Molecules, and Diseases, Vol. 55, No. 3, 01.10.2015, p. 266-271.

Research output: Contribution to journalArticle

Sega, Martiana Florenta ; Chu, Haiyan ; Christian, John A. ; Low, Philip S. / Fluorescence assay of the interaction between hemoglobin and the cytoplasmic domain of erythrocyte membrane band 3. In: Blood Cells, Molecules, and Diseases. 2015 ; Vol. 55, No. 3. pp. 266-271.
@article{a8bf5871d533472786c6c80e6022d8cc,
title = "Fluorescence assay of the interaction between hemoglobin and the cytoplasmic domain of erythrocyte membrane band 3",
abstract = "Oxygen tension has emerged as a potent regulator of multiple erythrocyte properties, including glucose metabolism, cell volume, ATP release, and cytoskeletal organization. Because hemoglobin (Hb)1 binds to the cytoplasmic domain of band 3 (cdb3) in an oxygen dependent manner, with deoxyHb exhibiting significantly greater affinity for cdb3 than oxyHb, the deoxyHb-cdb3 interaction has been hypothesized to constitute the molecular switch for all O2-controlled erythrocyte processes. In this study, we describe a rapid and accurate method for quantitating the interaction of deoxyHb binding to cdb3. For this purpose, enhanced green fluorescent protein (eGFP) is fused to the COOH-terminus of cdb3, and the binding of Hb to the NH2-terminus of cdb3-eGFP is quantitated by Hb-mediated quenching of cdb3-eGFP fluorescence. As expected, the intensity of cdb3-eGFP fluorescence decreases only slightly following addition of oxyHb. However, upon deoxygenation of the same Hb-cdb3 solution, the fluorescence decreases dramatically (i.e. confirming that deoxyHb exhibits much greater affinity for cdb3 than oxyHb). Using this fluorescence quenching method, we not only confirm previously established characteristics of the Hb-cdb3 interaction, but also establish an assay that can be exploited to screen for inhibitors of the sickle Hb-cdb3 interaction that accelerates sickle Hb polymerization.",
keywords = "Anion exchanger 1, FRET analysis, Hemoglobin, Protein structure, Red blood cells",
author = "Sega, {Martiana Florenta} and Haiyan Chu and Christian, {John A.} and Low, {Philip S.}",
year = "2015",
month = "10",
day = "1",
doi = "10.1016/j.bcmd.2015.07.004",
language = "English (US)",
volume = "55",
pages = "266--271",
journal = "Blood Cells, Molecules, and Diseases",
issn = "1079-9796",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - Fluorescence assay of the interaction between hemoglobin and the cytoplasmic domain of erythrocyte membrane band 3

AU - Sega, Martiana Florenta

AU - Chu, Haiyan

AU - Christian, John A.

AU - Low, Philip S.

PY - 2015/10/1

Y1 - 2015/10/1

N2 - Oxygen tension has emerged as a potent regulator of multiple erythrocyte properties, including glucose metabolism, cell volume, ATP release, and cytoskeletal organization. Because hemoglobin (Hb)1 binds to the cytoplasmic domain of band 3 (cdb3) in an oxygen dependent manner, with deoxyHb exhibiting significantly greater affinity for cdb3 than oxyHb, the deoxyHb-cdb3 interaction has been hypothesized to constitute the molecular switch for all O2-controlled erythrocyte processes. In this study, we describe a rapid and accurate method for quantitating the interaction of deoxyHb binding to cdb3. For this purpose, enhanced green fluorescent protein (eGFP) is fused to the COOH-terminus of cdb3, and the binding of Hb to the NH2-terminus of cdb3-eGFP is quantitated by Hb-mediated quenching of cdb3-eGFP fluorescence. As expected, the intensity of cdb3-eGFP fluorescence decreases only slightly following addition of oxyHb. However, upon deoxygenation of the same Hb-cdb3 solution, the fluorescence decreases dramatically (i.e. confirming that deoxyHb exhibits much greater affinity for cdb3 than oxyHb). Using this fluorescence quenching method, we not only confirm previously established characteristics of the Hb-cdb3 interaction, but also establish an assay that can be exploited to screen for inhibitors of the sickle Hb-cdb3 interaction that accelerates sickle Hb polymerization.

AB - Oxygen tension has emerged as a potent regulator of multiple erythrocyte properties, including glucose metabolism, cell volume, ATP release, and cytoskeletal organization. Because hemoglobin (Hb)1 binds to the cytoplasmic domain of band 3 (cdb3) in an oxygen dependent manner, with deoxyHb exhibiting significantly greater affinity for cdb3 than oxyHb, the deoxyHb-cdb3 interaction has been hypothesized to constitute the molecular switch for all O2-controlled erythrocyte processes. In this study, we describe a rapid and accurate method for quantitating the interaction of deoxyHb binding to cdb3. For this purpose, enhanced green fluorescent protein (eGFP) is fused to the COOH-terminus of cdb3, and the binding of Hb to the NH2-terminus of cdb3-eGFP is quantitated by Hb-mediated quenching of cdb3-eGFP fluorescence. As expected, the intensity of cdb3-eGFP fluorescence decreases only slightly following addition of oxyHb. However, upon deoxygenation of the same Hb-cdb3 solution, the fluorescence decreases dramatically (i.e. confirming that deoxyHb exhibits much greater affinity for cdb3 than oxyHb). Using this fluorescence quenching method, we not only confirm previously established characteristics of the Hb-cdb3 interaction, but also establish an assay that can be exploited to screen for inhibitors of the sickle Hb-cdb3 interaction that accelerates sickle Hb polymerization.

KW - Anion exchanger 1

KW - FRET analysis

KW - Hemoglobin

KW - Protein structure

KW - Red blood cells

UR - http://www.scopus.com/inward/record.url?scp=84937900247&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84937900247&partnerID=8YFLogxK

U2 - 10.1016/j.bcmd.2015.07.004

DO - 10.1016/j.bcmd.2015.07.004

M3 - Article

VL - 55

SP - 266

EP - 271

JO - Blood Cells, Molecules, and Diseases

JF - Blood Cells, Molecules, and Diseases

SN - 1079-9796

IS - 3

ER -